The expression of polypeptides or proteins, by recombinant DNA methods is now possible by fairly well established methods. Briefly, it consists of cloning the gene encoding a protein of interest in a suitable heterlogous host such as E. coli, yeasts or mammalian cells and culturing the “recombinant” hosts in suitable culture conditions to induce the expression of the protein. The protein so expressed may either be retained intracellularly, or be secreted into the extracellular medium. The protein is then isolated and purified from the culture medium.
There are several methods available for the isolation and purification of proteins. Typically, when a protein is expressed, the culture medium containing the expressed protein is first “clarified” to get rid of the nonessential particulate components—cells and/or cell debris. This is then followed by one or more chromatographic procedures in which the particular protein is isolated from the clarified medium, the choice of the procedure depending upon the nature and properties of the protein to be purified.
The clarification of the culture medium, containing the expressed protein, is usually carried out by either centrifugation or filtration. But both these methods are greatly limited in terms of efficiency of clarification and the volume of culture medium that can be handled at any given time. More often then not, both these methods need to be employed, with the centrifugation step usually preceding the filtration process. This in turn adds considerably to the cost of the overall process as well as increase the processing time, the latter increasing the risk of protein degradation and hence product loss.
An important consideration during the isolation of proteins from the medium of expressing cells is the actual availability of the proteins in soluble form in the medium. Whether secreted into the extracellular medium, or retained inside the host cell, a considerable portion of the expressed protein may remain bound to the cells and cellular debris. Following medium clarification, these “bound” fractions would remain in the particulate residue and would be lost from the filtrate/supernatant that is being subjected to subsequent chromatographic steps. Thus the final yield of the protein would be much less then had the residue been subjected to separate extraction steps to extract the bound protein into solution. On the other hand, separate “extraction” steps, with different solvents, additives or pH conditions, followed by conventional clarification and chromatography would add to the overall number of “processing” steps, increasing the process time (and the consequent product degradation and loss) and costs of protein extraction and purification.
Thus it would be highly desirable if the steps of extraction, medium clarification and chromatography could be combined to isolate and purify an expressed protein. So far, however, most of the prior art dealing with protein purification procedures generally describe the more conventional chromatographic methods for protein isolation and purification.
For example, U.S. Pat. No. 4,616,078 and EP0197764 describe a process for separating impurities from “impure mixtures” containing proinsulin like material. The process consists of applying the mixture to a reverse phase macroporous acrylate ester copolymer resin and eluting the proinsulin like material under specific pH and buffer conditions.
Processes for the separation and isolation of basic protein from protein mixture, employing strong acid cation exchange chromatography and consisting of elution buffers containing a small amount of alkanol is described in U.S. Pat. No. 5,101,013 and EP0305760.
U.S. Pat. No. 5,245,008 and EP0474213 describe a process for the purification of insulin and/or insulin derivatives on lipophilically modified silica gel, employing aqueous, buffered solvents containing zwitterions, with the pH of the buffer in the vicinity of the isoelectric point of the insulin/insulin derivative.
U.S. Pat. No. 5,621,073 and EP0547544 describe a process for obtaining insulin, free of any acylated forms and proteases, by chromatography on a lipophilically modified silica gel.
Methods for improved separation and isolation of insulin from enzymatic cleavage reaction, using pressure stable acidic cation exchange resins are described in U.S. Pat. No. 5,977,297 and EP0849277.
U.S. Pat. No. 6,451,987 and EP1163261 describe various ion exchange chromatography methods employing an elution technique, which is a combination of elution in a solution comprising an organic modifier (such as C1-6-alkanol, C1-6 alkenol, urea, guanidine etc.) with subsequent elution in an aqueous solution.
U.S. Pat. No. 6,398,963, U.S. Pat. No. 6,428,707, EP0538467, WO9218237, WO9833572 and EP1003599 describe affinity based gel matrices, and methods for adsorption of a substance from a liquid sample, on an expanded bed mode. Finally, the paper of Scot Shepard, Gregory Boyd and Jeffrey Schrimsher (Bioseparation Vol 10 Pages 51-56), 2001 describe the use strong cation exchange expanded bed adsorption chromatography for the purification of two proteins, one with 183 amino acids and the other with 260 amino acids.
While some these patents describe methods for the isolation of insulin (and proteins in general) present in solution, they do not provide methods for isolating insulin that remain bound to cellular surfaces and debris of expressing cells. The processes described in most of the above patents presuppose the requirement of filtration/centrifugation, to get rid of the particulate residue, prior to chromatography of the clarified medium. Thus any bound insulin (or protein) would either be lost, or separate extraction of the particulate residue would be needed. As mentioned above, this would increase the number of processing steps, and the time required for isolation and extraction of the protein. On the other hand, the paper by Shepard et al. merely describes an expanded bed adsorption cation exchange chromatography of two proteins of 183 and 260 amino acid size, while the U.S. Pat. Nos. 6,398,963 and 6,428,707 describe the use of expanded bed chromatography (but in the form of affinity chromatography). There is no description of ways to effect the simultaneous isolation of “in solution”, as well as particulate-residue-bound insulin. In the present invention, we disclose conditions for the extraction of insulin, to increase its recovery in solution, viz., in the liquid/buffer/medium in which the insulin expressing yeast culture has been suspended; and a procedure to combine extraction, medium clarification and chromatography, to effect the simultaneous isolation and purification of “in solution” as well as “particulate-residue-bound insulin”. By insulin we mean “native” human insulin or the “native” insulin of non-human origin, such as those of porcine and bovine origin. The term “insulin” could also include “pro”insulins, “prepro”insulins, as well as insulin derivatives including those modified chemically and enzymatically. In addition, “insulin” could also include analogs with changes in the primary sequence. The analogues could be analogues of human insulin, as well as non-human insulin, such as porcine and bovine insulin.